Inbred maize line R327H

ABSTRACT

The present invention is drawn to a novel DNA construct comprising an expression cassette having a constitutive promoter which functions in plant cells operably linked to a maize alcohol dehydrogenase intron, a DNA sequence of a gene encoding a Cry 1Ab protein, and a terminator functional in plants and optionally further comprising a second cassette including a promoter which functions in plants operably linked to a maize alcohol dehydrogenase intron, a DNA sequence of a gene encoding for phosphinothricin acetyl transferase, and a terminator functional in plants wherein the two cassettes are transcribed in the same direction. Also provided are transgenic plants, particularly maize plants, having such a construct stably incorporated into their genomes.

This application is a continuation of U.S. application Ser. No.09/042,426, filed Mar. 13, 1998, U.S. Pat. No. 6,114,608 the contents ofwhich are incorporated herein by reference, which claims the benefits ofU.S. application No. 60/109,808, filed Mar. 14, 1997 ABN., initiallyfiled as a regular U.S. application and subsequently converted to aprovisional U.S. application, the contents of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

This invention relates to a novel promoter, a novel DNA constructcontaining the promoter and a Bt gene, and plants, especially cornplants, containing the novel DNA construct.

Bacillus thuringiensis (Bt) belongs to a large group of gram-positive,aerobic, endospore forming bacteria. During sporulation, these specificbacteria produce a parasporal inclusion body which is composed ofinsecticidally active crystalline protoxins, also referred to asδ-endotoxins.

These endotoxins are responsible for the toxicity of Bacillusthuringiensis to insects. The endotoxins of the various Bacillusthuringiensis strains are characterized by high specificity with respectto target organisms. With the introduction of genetic engineering it hasbecome possible to create recombinant Bt strains which may contain achosen array of insect toxin genes, thereby enhancing the degree ofinsecticidal activity against a particular insect pest.

The insecticidal crystal proteins from Bt have been classified basedupon their spectrum of activity and sequence similarity (Hofte andWhiteley, Microbiol. Rev., 1989, 53:242-255 and Yamamoto and Powell,Advanced Engineered Pesticides, 1993, 3-42). Hofte and Whiteleypublished a classification scheme for the cry genes. Type I genes wereconsidered active only against Lepidoptera species; Type II genes wereactive against Lepidoptera and Diptera species; Type III genes wereactive against Coleoptera species and Type IV genes included both 70-and 130-kDa crystal protein and were highly active against mosquito andblackfly larvae. However, since this original classification many novelcry genes have been cloned and sequenced demonstrating that the originalsystem based on insect specificity required modification. Aclassification based on sequence homology along with new nomenclaturebased solely on amino acid identity has been proposed. (See Crickmore etal., Abstracts 28th Ann. Meeting Soc. Invert. Path. (1995), p14, Soc.Invert. Path., Bethesda Md.).

In this invention, the Cry proteins which are particularly effectiveagainst Lepidoptera species are preferred. These proteins are encoded bythe following nonlimiting group of genes: cry1Aa, cry1Ab, cry1Ac, cry1B,cry1C, cry1D, cry1E, cry1F, cry1G, cry2A, cry1C, cry5 and fusionproteins thereof. Among the cry genes, cry1Aa, cry1Ab, and cry1Ac showmore than 80% amino acid identity and cry1Ab appears to be one of themost widely distributed cry genes. The Cry1Ab proteins are particularlyeffective against larvae of Lepidoptera (moths and butterflies).

The ingestion of these proteins, and in some cases the spores, by thetarget insect is a prerequisite for insecticidal activity. The proteinsare solubilized in the alkaline conditions of the insect gut andproteolytically cleaved to form core fragments which are toxic to theinsect. The core fragment specifically damages the cells of the midgutlining, affecting the osmotic balance. The cells swell and lyse, leadingto eventual death of the insect.

A specific Lepidoptera insect, Ostrinia nubilalis (European corn borer(ECB)), causes significant yearly decrease in corn yield in NorthAmerica. One study reveales that approximately 10% of the corn acresplanted in the State of Illinois experienced a 9 to 15 percent annualyield loss, attributable solely to damage caused by the secondgeneration of corn borer. Other important lepidopteran insect pests ofcorn include Diatraea grandiosella (Southwestern Corn Borer),Helicoverpa zea (Corn Earworm) and Spodoptera frugiperda (FallArmyworm). The management practices of planting resistant or tolerantcorn hybrids and treatment with chemical and microbial insecticides havenot been satisfactory due to the low level of control provided byinsecticidal treatments and the lack of hybrid lines resistant to secondgeneration corn borers. Further tolerant and resistant hybrids often donot yield as well when infestation of ECBs are heavy. The use of corngenetically engineered to be resistant to specific corn insect pests hasmany advantages and these include a potential for substantial reductionin chemical insecticides and selective activity of the engineeredendotoxin which will not disrupt the population of beneficial non-targetinsect and animals.

Toxic Bt genes from several subspecies of Bt have been cloned andrecombinant clones have been found to be toxic to lepidopteran, dipteranand coleopteran insect larvae. However, in general, the expression offull length lepidopteran specific Bt genes has been less thansatisfactory in transgenic plants (Vaeck et al, 1987 and Barton et al,1987). It has been reported that the truncated gene from Bt kurstaki maylead to a higher frequency of insecticidal control. (U.S. Pat. No.5,500,365). Modification of the existing coding sequence by inclusion ofplant preferred codons including removal of ATTTA sequences andpolyadenylation signals has increase expression of the toxin proteins inplants. (U.S. Pat. No. 5,500,365). In the present invention a truncatedBt kurstaki HD-1 gene has been used.

The instant invention additionally includes a second coding segment. Thesecond coding segment comprises a DNA sequence encoding a selectivemarker for example, antibiotic or herbicide resistance including cat(chloramphenicol acetyl transferase), npt II (neomycinphosphototransferase II), PAT (phosphinothricin acetyltransferase), ALS(acetolactate synthetase), EPSPS (5-enolpyruvyl-shikimate-3-phosphatesynthase), and bxn (bromoxynil-specific nitrilase). A preferred markersequence is a DNA sequence encoding a selective marker for herbicideresistance and most particularly a protein having enzymatic activitycapable of inactivating or neutralizing herbicidal inhibitors ofglutamine synthetase. The non-selective herbicide known as glufosinate(BASTA® or LIBERTY®) is an inhibitor of the enzyme glutamine synthetase.It has been found that naturally occurring genes or synthetic genes canencode the enzyme phosphinothricin acetyl transferase (PAT) responsiblefor the inactivation of the herbicide. Such genes have been isolatedfrom Streptomyces. These genes including those that have been isolatedor synthesized are also frequently referred to as bar genes. As usedherein the terms “bar gene” and “pat gene” are used interchangeably.These genes have been cloned and modified for transformation andexpression in plants (EPA 469 273 and U.S. Pat. No. 5,561,236). Throughthe incorporation of the pat gene, corn plants and their offspring canbecome resistant against phosphinothricin (glufosinate).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents a plasmid map of pZO960 which contains the Bt kurstakiexpression cassette.

FIG. 2 represents a plasmid map of the base transformation vector pZO997

FIG. 3 represents a plasmid map of pZO1500 which contains the PATcassette.

FIG. 4 represents a plasmid map of the (expression/transformation)vector pZO1502 which contains the Bt kurstaki cassette and the PATcassette.

SUMMARY OF THE INVENTION

The present invention is drawn to a novel recombinant DNA constructcomprising an expression cassette includes a constitutive promoter whichfunctions in plant cells operably linked to an intron that functions inmonocots; a DNA sequence of a gene encoding an insecticidal Bacillusthuringiensis protein toxin; and a terminator functional in plants; andoptionally further comprises a second cassette which includes a promoterwhich functions in plant cells operably linked to an intron thatfunctions in monocots; a DNA sequence of a gene encoding forphosphinothricin acetyl transferase; and a terminator functional inplants, wherein the two cassettes are transcribed in the same direction.

Therefore a first aspect of the present invention is a DNA constructwhich expresses the crystal protein toxin of a Bt effective againstLepidopteran insects at relatively high levels and further providesresistance to the non-selective herbicide glufosinate.

A second aspect of the invention is a plant transformation vectorcomprising the DNA construct as given above.

A third aspect of the present invention comprises a transformed plantcell including the DNA construct as given above wherein the DNA isstably incorporated in the plant genome.

A fourth aspect of the invention is a plant comprising transformed plantcells wherein the DNA construct as given above is stably incorporatedinto the genome of the plant.

The invention further encompasses plant seeds having the DNA constructas given above stably incorporated therein.

Another aspect of the invention includes a plant cell co-transformedwith a first nucleic acid construct comprising, a CaMV 35S constitutivepromoter which functions in plant cells operably linked to a maizealcohol dehydrogenase intron, a DNA sequence of a gene encoding a Cry1Abprotein toxin or a functionally related protein toxin, and a terminatorfunctional in plants and a second nucleic acid construct comprising aCaMV 35S promoter which functions in plant cells operably linked to amaize alcohol dehydrogenase intron, a DNA sequence of a gene encodingfor phosphinothricin acetyl transferase, and a terminator functional inplants wherein the first and second constructs are stably integrated inthe plant genome.

The DNA construct of the invention preferably is an expression cassettefunctional in a plant comprising a promoter functional in plants, forexample a CaMV 35S promoter, e.g., as disclosed in SEQ ID No. 1 or 5,preferably SEQ ID No. 1, operably linked to an intron which functions inmonocots, for example a maize alcohol dehydrogenase intron, e.g., asdisclosed in SEQ ID No. 2 or 6, preferably SEQ ID No. 2. Thispromoter/intron sequence is operably linked to a DNA sequence ofinterest, for example a gene encoding a Bt delta-δ-endotoxin, e.g.,encoding the toxin domain of a Cry 1Ab protein or a functionally relatedtoxin protein, preferably modified for expression in plants, for exampleas depicted in SEQ ID No. 3, or a gene for a selectable marker, forexample a gene for herbicide resistance, preferably glufosinateresistance, for example a Pat gene, e.g., as depicted in SEQ ID No. 7.The gene of interest is suitably linked to a terminator functional inplants, e.g. a Nos terminator, for example as disclosed in SEQ ID No. 4or 8, preferably SEQ ID No. 4, to form an expression cassette functionalin a plant. Especially preferred embodiments of the Bt expressioncassette comprise SEQ ID Nos. 1, 2, 3 and 4 in operable sequence, e.g.,as in the Btk cassette described below. Especially preferred embodimentsof a Pat expression cassette comprise SEQ ID Nos. 5, 6, 7, and 8 inoperable sequence. In an especially preferred embodiment, a Btexpression cassette as described herein is linked on the same DNA with aPat expression cassette as described herein, e.g., a plasmid comprisingcassettes formed by SEQ ID Nos. 1-4 and 5-8 wherein the two cassettesare transcribed in the same direction, e.g., as in plasmid pZO1502.

The use of such expression cassettes in a method of transforming plants,e.g., maize plants, for example a method or biolistic or protoplasttransformation of maize plants, especially protoplast transformation asdescribed in the examples herein is also provided, as are plants stablytransformed with expression cassettes as described, particularly maizeplants, e.g., field corn, sweet corn, white corn, silage corn andpopcorn, and seed thereof. Particularly preferred are maize plants andseed thereof descended from the Bt11 transformation event described inExample 2, for example

Maize containing the Btk construct described within a 15 cM region ofchromosome 8, near position 117, in the approximate position of publicprobe UMC30a, in the interval flanked by two markers: Z1B3 and UMC150a,preferably

(i) elite inbred sweet corn lines R327H, R372H, R412H, R583H and R660H,

(ii) elite inbred field corn lines 2043Bt, 2044Bt, 2070Bt, 2100Bt,2114Bt, 2123Bt, 2227Bt, 2184Bt, 2124Bt, and 2221Bt, and

(iii) maize inbred varieties descended from the same transgenic event asthese lines which contain and express the same transgenic construct,

including seed thereof.

When particular inbred varieties are identified herein, it is understoodthat the named varieties include varieties which have the same genotypicand phenotypic characteristics as the identified varieties, i.e., arederived from a common inbred source, even if differently named. Theinvention also provides hybrid maize seed produced by crossing plants ofan inbred corn line as described above with plants of having a differentgenotype, and hybrid corn plants produced by growing such hybrid maizeseed. Also provided is a method of producing hybrid maize seedscomprising the following steps:

A. planting in pollinating proximity seeds of a first inbred maize lineas described herein and seeds of a second inbred line having a differentgenotype;

B. cultivating maize plants resulting from said planting until time offlowering;

C. emasculating said flowers of plants of one of the maize inbred lines;

D. allowing pollination of the other inbred line to occur, and

E. harvesting the hybrid seeds produced thereby.

Also provided are hybrid seeds produced by this method, F1 hybrid plantsproduced by growing such seeds, and parts of such F1 hybrid plants,including seeds thereof.

Seeds of the plants described herein (e.g., of maize plants, e.g., Bt11maize plants, for example inbred or hybrid seeds as described above) forplanting purposes is preferably containerized, e.g., placed in a bag orother container for ease of handling and transport and is preferablycoated, e.g., with protective agents, e.g., safening or pesticidalagents, in particular antifungal agents and/or insecticidal agents. Oneparticular embodiment of this invention is isolated inbred seed of theplants described herein, e.g. substantially free from hybrid seed orseed of other inbred seed, e.g., a seed lot or unit of inbred seed whichis at least 95% homogeneous, e.g., isolated seed of any of the maizeinbreds described in example 8 or 9 hereof.

Also provided herein, for the first time, are Bt maize varieties otherthan Bt field corn, particularly Bt sweet corn. Although Bt field cornhas been disclosed, it was not previously determined experimentallywhether or how a Bt delta δ-endotoxin would interact with traitsassociated with sweet corn, which is harvested at an earlier maturity(before it is dry), for a different purpose (usually fresh produce,canning or freezing, for human consumption) and has been bred thereforeto be qualitatively and quantitatively different from field corn in anumber of respects. Therefore, in one embodiment, the inventioncomprises a sweet corn comprising in its genome an expression cassettecomprising a coding region for a Bt delta-δ-endotoxin or functionalfragment or derivative thereof, under control of a promoter operable inmaize, e.g., an expression cassette as described herein. The sweet cornof the invention includes sweet or supersweet maize having a highersugar to starch ratio than field corn (e.g., yellow dent corn) due to areduced capacity to convert sugar into starch, typically characterizedby a sugary (su, e.g., su1) allele in the case of sweet corn, and/orshrunken allele (sh, e.g., sh2) or brittle allele (bt, e.g., bt2, not tobe confused with the gene for an endoxin from Bacillus thuringiensis,described elsewhere herein) in the case of supersweet corn, especiallymaize containing the su1 or sh2 alleles.

Bt maize of the invention, e.g., Bt11 maize, is found to be particularlysuited for the preparation of food materials (e.g., for human or animalconsumption, for example sweet corn for for packaging or fresh use as ahuman food, or grain or silage made from field corn) containing reducedlevels of fungal toxins, e.g., aflatoxins. While the mechanism is notentirely understood, in grain and silage made from Bt11 maize, the levelof aflatoxin is believed to be lower, possibly because the reduction ininsect damage reduces the level of opportunistic fungal infection in thegrowing plant. Accordingly, food materials made from Bt maize of theinvention, particularly Bt11 maize, for example grain and silage havingreduced levels of fungal toxins, particularly aflatoxins, and the use ofthe Bt maize of the invention in a method of preparing a foodstuff,especially grain or silage, with reduced levels of fungal toxins, e.g.,aflatoxins, is also provided.

DETAILED DESCRIPTION OF THE INVENTION

A promoter is defined as a nucleotide sequence at the 5′ end of astructural gene which directs the initiation of transcription. Thestructural gene is placed under regulatory control of the promoter.Various promoters which are active in plant cells are known anddescribed in the art. These include Cauliflower Mosaic Virus (CaMV) 19Sand 35S; nopaline synthase (NOS); mannopine synthase (MAS); actin;ubiquitin; ZRP; chlorophyll AB binding protein (CAB); ribulosebisphosphate carboxylase (RUBISCO); heat shock Brassica promoter (HSP80); and octopine synthase (OSC). The particular promoter used in thepresent invention should be capable of causing sufficient expression toresult in production of an effective amount of protein. The promoterused in the invention may be modified to affect control characteristicsand further may be a composite of segments derived from more than onesource, naturally occurring or synthetic. The preferred promoters areCaMV promoters and particularly CaMV 35S. The term “CaMV 35S” includesvariations of the promoter wherein the promoter may be truncated oraltered to include enhancer sequences, to increase gene expressionlevel, and composite or chimeric promoters, wherein portions of anotherpromoter may be ligated onto the CaMV 35S. A preferred embodimentincludes the 5′ untranslated region of the native 35S transcript, andmore particularly wherein the untranslated region includes about 100 to150 nucleotides. Additionally while 35S promoters are fairly homologous,any 35 S promoter in a preferred embodiment would include theuntranslated region of the native 35S transcript. Particularly preferred35S promoters are described in SEQ ID NO. 1 and SEQ ID NO. 5. Thepromoter as described in SEQ ID NO. 1 as part of the claimed constructmay have particular advantage in that the construct may be expressed inpollen tissue.

An intron is a transcribed nucleotide sequence that is removed from theRNA transcript in the nucleus and is not found in the mature mRNA. Suchsequences are well known in the art, and monocot introns include but arenot limited to sucrose synthetase (SS); glutathione transferase; actin;and maize alcohol dehydrogenase introns. An exon is part of a gene thatis transcribed into a mRNA and includes non-coding leader and/or trailersequences. An exon may code for a specific domain of a protein. Havingnative exon sequences around an intron may improve the introns splicingactivity or the ability of the nuclear splicesomal system to properlyrecognize and remove the intron. According to the invention, a preferredembodiment includes the native exon in the first cassette and moreparticularly 50 or more nucleotide bases of the native exon on each sideof the intron is preferred.

A gene refers to the entire DNA sequence involved in the synthesis of aprotein. The gene includes not only the structural or coding portion ofthe sequence but also contains a promoter region, the 3′ end and poly(A)sequences, introns and associated enhancers or regulatory sequences.

A structural heterologous gene is that part of a DNA segment whichencodes a protein, polypeptide or a portion thereof, and one which isnot normally found in the cell or in the cellular location where it isintroduced. The DNA sequence of a structural heterologous gene of thepresent invention include any DNA sequence encoding a crystal toxininsecticidal protein. The preferred toxins include but are not limitedto Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G,Cry2A, Cry2B, Cry3A, Cry3B, Cry3C, Cry4A, Cry4B, Cry4C, Cry4D, Cry5A,Cry9C, CytA and any fusion protein or truncated gene that encodes one ormore of the abovementioned toxins or a mixture thereof. Particularlypreferred toxins include Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry2A, Cry3C,Cry1E, Cry5A, Cry9C and any mixture or fusion protein thereof. In thepresent specification, the term fusion protein is used interchangeablywith the terms fusion toxin and hybrid protein and is a proteinconsisting of all or part of an amino acid sequence (known as a domain)of two or more proteins, and is formed by fusing the protein encodinggenes. An example of a DNA sequence useful in the cassette of thisinvention is a DNA sequence encoding a fusion toxin wherein the toxin isCry1Ab/Cry1C and Cry1E/Cry1C. The domains comprising the fusion proteinmay be derived from either naturally occurring or synthetic sources.

Many cry1Ab genes have been cloned and their nucleotide sequencesdetermined. A holotype gene sequence of cry1Ab has accession number M13898 (The GenBank v. 70/EMBL v.29). A number of studies reveal that theamino terminal end of the Cry1A protein is responsible for theinsecticidal activity. This region depends on the particular protein butin general include a truncated gene that encodes from about amino acid25 to amino acid 610 of the protein.

In the present invention, a preferred cry1Ab gene includes a syntheticgene encoding the toxin domain of the protein produced by the Btkurstaki (k) HD-1 gene wherein the G+C content of the Btk gene isincreased and the polyadenylation sites and ATTTA regions are decreased.U.S. Pat No. 5,500,365, which is hereby incorporated in its entiretydiscloses a synthetic Btk HD-1 and HD-73 gene, and truncated HD-1 andHD-73 genes. A particularly preferred cry1Ab gene of this invention isthe sequence as described in SEQ ID NO. 3.

Other preferred genes include those that are functionally equivalent tocry1Ab. These genes include all cry1Ab, cry1Aa, cry1Ac and variantsthereof wherein the expressed protein toxin is active against one ormore major maize Lepidoptera insect pests. The insect pests include theaforementioned European corn borer, Southwestern corn borer, Fallarmyworm, and Corn earworm.

The second structural gene that is part of the invention includes a DNAsequence encoding a selective marker for example, antibiotic orherbicide resistance including cat (chloramphenicol acetyl transferase),npt II (neomycin phosphototransferase II), PAT (phosphinothricinacetyltransferase), ALS (acetolactate synthetase), EPSPS(5-enolpyruvyl-shikimate-3-phosphate synthase), and bxn(bromoxynil-specific nitrilase). A preferred marker sequence is a DNAsequence encoding a selective marker for herbicide resistance and mostparticuarly a protein having enzymatic activity capable of inactivatingor neutralizing herbicidal inhibitors of glutamine synthetase. Thenon-selective herbicide known as glufosinate (BASTA® or LIBERTY®) is aninhibitor of the enzyme glutamine synthetase. It has been found thatnaturally occurring genes or synthetic genes can encode the enzymephosphinothricin acetyl transferase (PAT) responsible for theinactivation of the herbicide. Such genes have been isolated fromStreptomyces. Specific species include Streptomyces hygroscopicus(Thompson C. J. et al., EMBO J., vol. 6:2519-2523 (1987)), Streptomycescoelicolor (Bedford et al, Gene 104: 39-45 (1991)) and Streptomycesviridochromogenes (Wohlleben et al. Gene 80:25-57 (1988)). These genesincluding those that have been isolated or synthesized are alsofrequently referred to as bar genes. As used herein the terms “bar gene”and “pat gene” are used interchangeably. These genes have been clonedand modified for transformation and expression in plants (EPA 469 273and U.S. Pat. No. 5,561,236). Through the incorporation of the pat gene,corn plants and their offspring can become resistant againstphosphinothricin (glufosinate). A preferred coding segment of a bar geneof the present invention is the sequence described in SEQ ID NO. 7.

The structural gene of this invention may include one or moremodifications in either the coding region or in the untranslated regionwhich would not substantially effect the biological activity or thechemical structure of the expression product, the rate of expression orthe manner of expression. These modifications include but are notlimited to insertions, deletions, and substitutions of one or morenucleotides, and mutations. The term homology as used hereinrefers toidentity or near identity of nucleotide or amino acid sequences. Theextent of homology is often measured in terms of percentage of identitybetween the sequences being compared. It is understood in the art thatmodification can occur in genes and that nucleotide mismatches and minornucleotide modifications can be tolerated and considered insignificantif the changes do not alter functionality of the final product. As inwell known in the art the various cry1A genes have very similar identityand reference in made to the article by Yamamoto and Powell, AdvancedEngineered Pesticides, 1993, 3-42 which includes a dendrogram tableshowing sequence homology among full length crystal proteins obtainedfrom the GenBank data base for a full length comparision.

Termination sequences are sequences at the end of a transcription unitthat signals termination of transcription. Terminators are 3′non-translated DNA sequences that contain a polyadenylated signal.Examples of terminators are known and described in the literature. Theseinclude but are not limited to nopline synthase terminator (NOS); the35S terminator of CaMV and the zein terminator.

Other elements may be introduced into the construct for examples matrixattachments region elements (MAR). These elements can be positionedaround an expressible gene of interest to effect an increase in overallexpression of the gene and to diminish position dependent effects uponincorporation into the plant genome.

Transformation means the stable integration of a DNA segment carryingthe structural heterologous gene into the genome of a plant that did notpreviously contain that gene. Co-transformation is transformation withtwo or more DNA molecules. Frequently one segment contains a selectablegene generally one for antibiotic or herbicide resistance.

As used herein the term plant tissue is used in a wide sense and refersto differentiated and undifferentiated plant tissue including but notlimited to, protoplasts, shoots, leaves, roots, pollen, seeds, callustissue, embryos, and plant cells (including those growing or solidifiedmedium or in suspension.

The DNA construct of this invention may be introduced into a planttissue by any number of art recognized ways. These included, but are notlimited to, direct transfer of DNA into whole cells, tissue orprotoplasts, optionally assisted by chemical or physical agents toincrease cell permeability to DNA, e.g. treatment with polyethyleneglycol, dextran sulfate, electroporation and ballistic implantation ofDNA coated particles. The following references further detail themethods available: Biolistic transformation or microprojectilebombardment (U.S. Pat. Nos. 4,945,050; 5,484,956; McCabe et al., AnnualRev. Genet. 22:421-477 (1988); Klein et al., Proc. Natl. Acad. Sci. USA,85:4305-4309 (1988); Klein et al., Bio/Technology 6:559-563 (1988);Gordon-Kamm et al., Plant Cell 2:603-618 (1990); and Vasil et al.,Bio/Technogy 11:1553-1558 (1993); Protoplast transformation—EPA 0 292435; EPA 0 465 875; and U.S. Pat. NO. 5,350,689; microinjection—Crosswayet al., BioTechniques 4: 320-334 (1986); direct gene transfer—Paszkoskiet al., EMBO J. 3:2717-2722 (1984); electrotransformation—U.S. Pat. No.5,371,003; and electroporation—Rigg et al., Proc. Natl, Acad. Sci. USA83: 5602-5606 (1986). Transformation is also mediated by Agrobacteriumstrains, notably A. tumefaciens and A. rhizogenes, and also by variousgenetically engineered transformation plasmids which include portions ofthe T-DNA of the tumor inducing plasmids of Agrobacteria. EPA 0 604662A1, Japan Tobacco Inc.; Hinchee et al., BioTechnology 6: 915-921(1988). Also see Potrykus, I. Annu. Rev. Plant Physiol. Plant Mol. Biol.1991, 42:205-225. The choice of a particular method may depend on thetype of plant targeted for transformation.

Transformed plants may be any plant and particularly corn, wheat,barley, sorghum, and rice plants, and more particularly corn plantsderived from a transformant or backcrossing through further breedingexperiments.

EXAMPLE 1

Plasmid Construction

A. Plasmid pZO1502 construction: The plasmid pZO1502 can be consideredto consist of three basic regions; the base plasmid vector, anexpression cassette for the Btk gene, and an expression cassette for thepat gene. For convenience, the various parts were constructed separatelyand then combined into the final plasmid. In order to assemble thedesired elements for the Btk and pat gene expression cassettes, therestriction sites used to generate the desired elements sometimesrequired modification. The following example demonstrates the procedureused to produce the pZO1502 plasmid. One skilled in the art could devisealternate ways to construct the final transformation plasmid.

B. Base Plasmid Vector: The base vector, pUC18 (GenBank accessionL08752, Norrander, J. M., et al., 1983. Gene 26:101-106), was modifiedby replacing the EcoO 109 I restriction site with a Bgl II linker(digestion with EcoO 109 I, fill in with T4 polymerase, and addition ofa Bgl II linker). This base vector was further modified to replace theBspH I sites at 1526 and 2534 with Not I restriction sites (vector cutwith BspH I, filled in, and replaced with Stu I linkers; the Stu I sitewas then cut and Not I linkers added). The addition of the Not Irestriction sites provided a convenient way to produce a linear DNAfragment containing the two desired gene cassettes (Btk and pat)separated from the ampicillin gene sequence (required for plasmidproduction in E. coli). This linearization also significantly increasedprotoplast transformation frequency. The final base vector was namedpZO997B (FIG. 2).

C: Btk gene expression cassette: The Dde I to Dde I fragment of the 35Spromoter from cauliflower mosaic virus (strain CM1841, GenBank accession# V00140, Gardner, R. C., et al., 1981. Nucleic Acids Res. 9:2871-2888)(SEQ ID NO. 1) was converted to Sac I by addition of linkers and clonedinto the Sac I site of the polylinker region of a pUC19 based vector.The sixth intron from maize Adh1-1S gene (GenBank accession X04049,Dennis, E. S., et al, 1984. Nucleic Acid Res. 12:3983-4000) was isolatedas a Pst I to Hpa II fragment, converted with BamH I linkers (SEQ. IDNO. 2), and cloned into the BamH I poly linker site 3′ to the 35Spromoter. The 3′ terminator from Nopaline synthetase, NOS, (GenBankaccession V00087, Bevan, M., et al., 1983. Nucleic Acids Res.11:369-385) (SEQ. ID NO 4) was isolated as ˜250 bp fragment with Pst Iand Bgl II. The Bgl II site was polished with T4 polymerase, a Hind IIIlinker added, and the fragment inserted behind a gus gene constructusing the Pst I and Hind III sites. The gus gene was cloned into the SalI to Pst I site of the polylinker. The gus construct utilized asynthetic linker (Sal I to Nco I, which provides for an A nucleotide atthe −3 position from the translation start ATG: GTCGACCATGG) (SEQ ID NO.9). The Pst I site was then trimmed, a Bcl I linker added, and the gusgene sequence replaced with a synthetic gene encoding a cry1Ab toxin(SEQ. ID NO. 3) as a Nco I to Bgl II insert to produce the vector pZO960(FIG. 1).

D. Pat gene expression cassette: Although composed of similar elements,the pat expression cassette was derived from a different series ofcloning steps. The 35S promoter (SEQ ID NO. 5) was obtained as a Hinc IIto Dde I fragment from the cauliflower mosaic virus (strain CABB-S,GenBank accession # V00141, Franck, A., et al., 1980. Cell 21: 285-294)and converted to BamH I-Xba I with linkers. The second intron sequencefrom maize Adh1-1S (GenBank accession X04049, Dennis, E. S., et al.,1984. Nucleic Acid Res. 12:3983-4000) (SEQ ID NO. 6) was isolated as aXho II to Xho II fragment and cloned into the BamH I site of pUC12,converting the Xho II sites to BamH I. As a BamH I fragment it wascloned into the Bgl II site of a synthetic polylinker (Asu II, Bgl II,and Xho I). The Asu II site was then filled in and ligated to the(filled in) Xba I site of the 35S promoter fragment. The synthetic patgene sequence was subcloned from plasmid pOAC/Ac (obtained from Dr.Peter Eckes, Massachusetts General Hospital, Boston Mass.) (SEQ ID NO.7) as a Sal I to Pst I fragment and combined with the 35S/Adhivs2promoter (Xho I) and the 3′ NOS terminator sequence Pst I to Bgl II(GenBank accession V00087, Bevan, M., et al., 1983. Nucleic Acids Res.11:369-385) (SEQ ID NO. 8). These pieces were all combined with thepZO997B base vector to produce the pat expression vector pZO1500 (FIG.3).

As the final construction step, the Btk expression cassette wassubcloned from pZO960 as an EcoR I-Hind III fragment and inserted intothe EcoR I-Hind III polylinker site of pZO1500 to produce the finalvector, pZO1502 (FIG. 4). The amp (beta-lactamase) gene was removedprior to plant transformation by digestion with NotI. pZO1502 has beendeposited with the American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, Md. 20852-1776 USA pursuant to the BudapestTreaty prior to the filing of this application and accorded accessionnumber 209682 on Mar. 13, 1998, and the complete sequence of thisplasmid is disclosed in SEQ. ID No. 9.

EXAMPLE 2

Protoplast Transformation, Selection of Transformed Corn Cells andRegeneration

The initial parental transformation of the corn line to be planted wasaccomplished through insertion of a DNA fragment from plasmid pZO1502,containing the two cassettes of Btk and the pat gene, into the genome ofa proprietary corn cell line owned by Hoerchst A G (Frankfurt Germany).The transformation was performed using a protoplast transformation andregeneration system as described in detail in European PatentApplication Publication No. 0 465 875 A, published Jan. 15, 1992 andEuropean Patent Application Publication No. 0 469 273 A, published Feb.5, 1992 and Theor. Appl. Gent. 80:721-726 (1990)). The contents of whichare hereby incorporated by reference.

After some weeks on selective media putative transformant clumps ofcells were observed and transformed protoplasts were selected in vitrowith a glufosinate-ammonium herbicide. Sixteen leaf producinggenetically transformed corn lines were obtained from protoplaststreated with the gene expression cassette from pZO1502. One of theselines was designated as transformant number 11. This transformant wasgrown to maturity.

The Bt-11 R0 transformed plants were pollinated with nontransformedNorthrup King elite inbred male parents and RI seed was collected.Descendants of the initial crossing have been successively backcrossedand test crossed to establish and evaluate corn lines carrying the Btkgene. Such lines are described more fully in the Examples 8 and 9 belowand have been deposited with the ATCC pursuant to the Budapest Treaty.

EXAMPLE 3

Stable Transformation

Expression of the Btk gene was tested by transforming the Bt gene vectorpZO960 into BMS (Black Mexican Sweet) corn cells. Protoplasts wereisolated from a suspension culture BMS cell line and electroporated toinduce DNA uptake essentially as described in Sinibaldi, R. M. andMettler, I. J., 1992, In: Progress in Nucleic Acid Research andMolecular Biology (W. E. Cohn and K. Moldave, eds.) Academic Press, SanDiego, vol. 42:229-259. Cells which had stably incorporated DNA wereselected by co-transformation with a plasmid containing a kanamycinresistance selectable gene. A number of independent transgenic eventswere selected by the expression of the antibiotic resistance tokanamycin. Approximately 1 gram of each transgenic line was then used totest for biological activity against neonate larvae of Manducca sexta.Control, non-transformed, BMS callus tissue supported normal growth ofthe larvae throughtout the test period. Transgenic callus lines werethen rated for the degree of growth inhibition. As shown in Table 1, outof 33 BMS lines co-transformed with pZO960, 6 lines were positive forinsecticidal activity showing complete growth inhibition and 100%mortality within 2 or three days. Quantitative Elisa assays showed thatthe transgenic tissues produced an average of 3.1 ng of Bt protein permg of total extracted protein.

TABLE 1 Stable Transformation with Btk Cassette Insect activity Bt ELISAassays Construct #pos/#test ng/mg protein pZO960 6⁺/33 3.1 + = stronginsecticidal activity, 100% mortality in 2-3 days, little feeding.

EXAMPLE 4

Insertion Site of Bt11 Transgenic Event

The original genetic stock into which the Btk sequence was transformedwas designated HE89. The Ro plants were used as the female parent forinitial crosses to two, elite Northrup King proprietary inbred lines forwhich Btk-conversion was sought. Multiple backcrosses were conductedinto many additional inbred lines with individuals selected thatcontained the insertion sequence but were, otherwise, as similar to theelite recurrent parents as possible. Four or more backcrosses andselfing to homozygosity were used in the conversion process. Finishedconversion stocks were evaluated with a series of 50 or 60 RFLP probesselected to be well distributed throughout the genome. Genotypes of theBtk converted inbreds were compared to those of their recurrent parentisolines. They were generally identical or nearly identical for allgenetic markers, except for three probes on a small segment of the longarm of chromosome 8. All conversion stocks differ from the genotype ofthe transformed stock, HE89, for this segment, thus differing from therecurrent parents. There were no other genomic regions with consistentdifferences between Btk-conversions and their recurrent parents. Thesethree probes exist within 10 centiMorgans(cM) of one another at theapproximate position of the public probe UMC30a, which has been placedat map position 117 in the 1995 map of RFLP probe positions distributedby the University of Missouri at Columbia.

A series of 95 backcross progeny were further characterized withnumerous probes in the region of chromosome 8 identified above. The sizeof the “donor” DNA segment varied among these progeny. However, five ofthe progeny failed to contain the donor alles at the flanking markers:Z1B3 and UMC150a, despite presence of the Btk sequence. These two probesare approximately 15 cM apart on chromosome 8. Thus, the insertion siteis within a 15 cM region on the long arm of chromosome 8, near position117, and in the interval flanked by two markers: Z1B3 and UMC150a.

Southern Analysis of the Transgenic Event

The Bt11 transgenic seeds backcrossed into inbred line HAF031 were sownin the greenhouse and sprayed with BASTA herbicide at the four leafstage. Resistant plants and control, untransformed , HAF031 inbredplants were then used for DNA extraction and Southern blot analysis (T.Maniatis, E. F. Fritsch and J. Sambrook, 1982, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory) The genomic DNAsamples were digested with following restriction enzymes and probed withlabeled DNA for Btk and PAT gene sequences. The first enzyme combinationutilized 2 restriction sites present on the plasmid DNA. The next twoenzymes had only one known location and would be expected to cut thegenomic DNA at a distant site in the plant DNA. The actual size of anyobserved fragment depends on the insertion event. The number of bandscan be used to estimate insertion copy number—each gene copy wouldproduce a unique band on the Southern blot.

The results of a Southern blot are summarized in Table 2. These datashow that the Bt11 transgenic lines are derived from a single insertionevent containing one gene copy of the Bt and pat gene sequences.

TABLE 2 Restriction Enzymes Probe Predicted - Observed #Fragment Sal 1and Sac I Btk 1.3 kb 1.3 kb 1 Hind III Btk >3 kb ˜30 kb 1 EcoR I Btk >5kb ˜25 kb 1 PstI and Hind III PAT 1.5 kb 1.5 kb 1 Hind III PAT >2 kb ˜30kb 1 EcoR I PAT >5 kb ˜25 kb 1 The DNA probe fragments were isolatedfrom the original plasmid vector pZO1502: Btk = Sal I and Sac I fragmentand PAT + Sal I fragment.

EXAMPLE 5

Enzymatic Activity of PAT in the Bt Transformed Lines

Fresh tissue samples (30-50 mg) were ground on ice in 5 volumes ofextraction buffer (100 mM Tris-HCL, pH 7.5), 3 mg/ml dithiothreitol and0.3 mg/ml bovine serum albumin (BAS fraction V). The homogenate wascentrifuged to clarity (12,000×g for 5 min). Approximately 2 μl ofextract was added to the reaction mixture containing the extractionbuffer plus 125 μM acetyl CoA and 250 μM phosphinothricin. The enzymaticreaction was allowed to proceed for 1 hour at 37° C. The reaction mixwas then spotted onto TLC silica gel plates (Baker Si250-PA (19C)). Theplate was chromatographed for 2-3 hours with isopropanol:NH4OH (3:2),air dried and vacuum dried in an oven at 80° C. The plates were thenexposed to X-ray film for 1-4 days. The results of a typical assayconfirm the presence and enzymatic activity of the PAT protein in the Btlines.

EXAMPLE 6

Inheritance and Gene Stability

The segregation of the Btk gene and the PAT gene were followed inmultiple generations. Eight F1 corn plants identified as containing theBtk and PAT genes were selfed to produce a S1 population. The S1population was screened for resistance to ECB and Ignite® herbicide. Allplants were either resistant to ECB and Ignite or susceptible to both.The segregation ratios were consistent with an expected ratio of 3:1 fora single dominant locus.

EXAMPLE 7

Bt-11 Maize Versus European Corn Borer Field Trials

Trials were conducted using a randomized complete block design. Tworeplicates were planted at three locations across three states intwo-row plots. Hybrids were grouped according to relative maturity andplanted at appropriate sites based on maturity. Southern trialscontained six Btk hybrids and four non-Btk control hybrids. The northerntrials consisted of eight Btk hybrids and two non-Btk hybrids. Plantswere artificially infected as they approached the V6 stage of growth.Approximately fifty larvae were applied to ten plants in the first rowof each plot every three to four days over a two and one-half weekperiod. By the end of the first generation infesting, each plant hadbeen infected with at least 200 neonate larvae. Just prior to tasselemergence, 1-9 leaf damage ratings were assigned to each of the tenplants per plot. The rating scale of Gurthie, W. D., et al. (1960, “Leafand Sheath Feeding Resistance to the European Corn Borer in Eight InbredLines of Dent Corn”, Ohio Ag. Exp, Sta. Res. Bull. 860) was used,wherein 1=no damage or few pinholes, 2=small holes on a few leaves,3=shot-holes on serval leaves, 4=irregular shaped holes on a few leaves,and 9=several leaves with many emerging elongated lesions.

As plants began to shed pollen, second generation ECB infestation began.The first ten plants of the first row of each plot were infected with40-50 larvae every three to four days over a two and one-half weekperiod. Eventually every plant had been infected with approximately 200more larvae. After approximately 45 to 50 days, plants were dissectedfrom top to the ground and the total length of tunnels created by ECBfeeding was estimated and converted to centimeters for reporting.Analysis of Variance and Least Significant Difference mean separationwere used to analyze the results.

Average leaf feeding damage scores were approximately 3.9 on non-Btkhybrids and 1.1 for Btk hybrids wherein 1 on the scale of 1 to 9represents no damage. Average stalk damage represented as centimeterstunneled per plant, was approximately 4.9 cm in the non-Btk controlhybrids. The Btk hybrids displayed only approximately 0.2 cm oftunneling per plant. In all cases, the difference between Btk hybridsand non-Btk hybrids was significant at a P-value of less than 0.01 basedon AVOVA and LSD mean separation. Field tests conducted to determinedthe resistance of Btk hybrids and non-Btk hybrids for Southwestern CornBorer and Fall Armyworm also indicated that Btk hybrids showed excellentpotential for assisting in the control of these insect pests.

EXAMPLE 8

Bt11 Sweet Corn

Inbred backcrossing of Bt11 event material as described in Example 4into Novartis (Rogers) elite inbred sweet corn lines was carried out toobtain Bt11 inbred sweet corn lines, including inbreds R327H, R372H,R412H, R583H and R660H. These inbreds and their F1hybrid progeny allcontain the Btk insert as described above at the location describedabove and exhibit insect resistance and herbicide resistance as for theother lines descended from the Bt11 event. For example, 2500 seeds ofeach of these lines were deposited with ATCC prior to the filing of thisapplication pursuant to the Budapest Treaty and accorded accessionnumbers as follows: R327H: ATCC Accession No:209673, deposited Mar. 11,1998, R372H: ATCC Accession No:209674, Mar. 11, 1998, R412H: ATCCAccession No:209675, deposited Mar. 11, 1998, R583H: ATCC AccessionNo:309671, deposited Mar. 11, 1998 and R660H: ATCC Accession No:209672,deposited Mar. 11, 1998. These lines were evaluated at Nampa, Id. andStanton, Minn. during the summer and fall of 1997, and characterized inrelation to a standard reference inbred (Iowa5125, from North CentralRegion Plant Introduction Center, Ames, Iowa) having similar backgroundand maturity, as depicted on the following table. (All measurements arein centimeters unless otherwise noted. Colors are according to Munsellcolor code chart.)

TABLE 3 Trait R327H R372H R412H R583H R660H Iowa5125 Kernel colorYellow- Yellow- Yellow- Yellow- Yellow- Yellow- orange orange orangeorange orange orange Endosperm type su1 su1 su1 sh2 sh2 su1 Maturity(days) emergence to 50% silk 71 70 75 70 77 71 emergence to 50% pollen68 67 68 66 73 67 50% silk to optimal edible 24 26 25 25 29 25 qualityPlant plant height 207.0 199.7 144.0 173.8 174.8 152.8 ear height 51.865.9 45.3 40.1 57.0 57.5 top ear internode 17.6 15.5 10.0 15.8 13.6 13.8avg. number of tillers 2.3 1.1 0.4 3.3 1.2 0.8 avg. number of ears/stalk1.8 1.9 1.7 2.1 2.0 1.3 anthocyanin of brace roots absent absent absentabsent absent absent Leaf width of ear node leaf 7.5 6.4 8.1 7.5 9.7 7.3length of ear node leaf 70.7 65.0 54.0 64.1 67.3 82.4 no. of leavesabove top ear 6 5 5 5 6 6 degrees of leaf angle 49 41 63 46 60 56 leafcolor very dark very dark green- very green- green- green green yellowdark yellow yellow green Tassel no. of primary lateral 15 9 16 10 16 28branches tassel length 45.8 42.0 31.0 41.6 34.5 28.4 Ear silk colorgreen- green- green- green- light light yellow yellow yellow yellowgreen green position at dry husk stage upright pendent horizontal —upright pendent ear length 14.5 16.0 15.3 16.7 15.7 13.3 ear diameter atmidpoint 4.1 3.8 3.74 4.67 4.05 5.33 number of kernel rows 16 16 16 1516 21 cob diameter at midpoint 2.59 2.50 2.53 2.61 2.54 2.94

EXAMPLE 9

Bt11 Field Corn

Inbred backcrossing of Bt11 event material as described in Example 4into Novartis (Rogers) elite inbred field corn lines was carried out toobtain Bt11 inbred field corn lines, for example Yellow Dent inbredlines 2044Bt, 2070Bt, 2100Bt, 2114Bt, 2123Bt, 2227Bt, 2184Bt, 2124Bt,and 2221Bt. These inbreds and their hybrid progeny all contain the Btkinsert as described above at the location described above and exhibitinsect resistance and herbicide resistance as for the other plantsdescended from the Bt I1 event. 2500 seeds of each of the followinglines were deposited with ATCC pursuant to the Budapest Treaty on Apr.19, 1999 and accorded deposit numbers as follows: 2044Bt: ATCC 203943,2070Bt: ATCC 203941 , 2227Bt: ATCC 203942, 2184Bt: ATCC 203944 and2221Bt:. Bt11 inbreds were also made by marker assisted inbredconversion of the following lines, NP948 (ATCC 209406), NP2017 (ATCC209543), NP904 (ATCC 209458), NP2010 (ATCC), all deposited with ATCCpursuant to the Budapest Treaty to obtain 2100Bt, 2114Bt, 2123Bt and2124Bt respectively.

Hybrids from Bt11 inbred conversions were evaluated extensively againsthybrids from isogenic, non-transgenic parents in a number of fieldtrials. In general, there was a significant yield advantage to the BT11version. There was no attempt to control natural infestations ofEuropean Corn Borers in these trial locations. Grain moisture at harvestis sometimes slightly higher in the BT11 version. This can often beattributed to the improved plant health, due to reduced stalk rot. Insome cases, grain test weight is higher in the BT11 version, which canalso reduce the rate of grain dry down. Stalk lodging is typically lowerin the BT11 versions. Push test and Late season intactness are alsotypically better in BT11 versions. In some cases, stay green is better.Plant and ear height are sometimes slightly higher in the BT11 version.For other traits, no consistent detrimental changes in performance havebeen observed. 2124Bt, 2221Bt, and 2070Bt are southern (late)maturities, whereas 2044Bt, 2100Bt, 2114Bt, 2227Bt, 2184Bt, and 2123Btare northern (early) maturities. These inbred Bt lines have thefollowing general characterization:

2044Bt—dark-reddish purple silk, slight pale green color, very slightlyfaded chlorotic stripes in leaves, medium tall, medium ear placement,purple tip to glume

2100Bt—green-yellow silk, medium-short plant height, medium low earplacement, green with purple glume, light green overall appearance

2114Bt—dark reddish purple silk, small tassel, slight crook in stalknodes, slight pale green color, medium tall, medium ear placement,higher yielding than 2044Bt

2227Bt—very thin loose husk at harvest, root lodges, medium plantheight, medium ear placement

2184Bt—medium plant height, medium ear placement, very light pollenshedder, green yellow silk color, pale purple anther

2123Bt—green with purple glumes, purple anther, green yellow silk,medium plant height

What is claimed is:
 1. Seed of maize inbred line R327H having beendeposited under ATCC Accession No:
 209673. 2. Seed according to claim 1,wherein said seed comprises a nucleic acid construct comprising twocassettes, wherein the first cassette comprises a CaMV 35S constitutivepromoter operably linked to a maize alcohol dehydrogenase intron, a DNAsequence of a gene encoding a Cry1Ab protein, and a terminatorfunctional in plants, and the second cassette comprises a CaMV 35Spromoter which functions in plant cells operably linked to a maizealcohol dehydrogenase intron, a DNA sequence of a gene encoding forphosphinothricin acetyl transferase, and a terminator functional inplants, wherein the two cassettes are transcribed in the same direction,wherein the nucleic acid construct is incorporated into the seed'sgenome on chromosome 8, near position 117, between markers Z1B3 andUMC150a.
 3. Seed according to claim 2, wherein the first expressioncassette comprises SEQ ID Nos. 1-4 in operable sequence.
 4. Seedaccording to claim 2, wherein the second expression cassette comprisesSEQ ID Nos. 5-8 in operable sequence.
 5. Seed according to claim 2,wherein the first expression cassette comprises SEQ ID Nos. 1-4 inoperable sequence and the second expression cassette comprises SEQ IDNos. 5-8 in operable sequence.
 6. A maize plant, or parts thereof, ofinbred line R327H, seed of said line having been deposited under ATCCaccession No:
 209673. 7. A maize plant according to claim 6, whereinsaid maize plant comprises a nucleic acid construct comprising twocassettes, wherein the first cassette comprises a CaMV 35S constitutivepromoter operably linked to a maize alcohol dehydrogenase intron, a DNAsequence of a gene encoding a Cry1Ab protein, and a terminatorfunctional in plants, and the second cassette comprises a CaMV 35Spromoter which functions in plant cells operably linked to a maizealcohol dehydrogenase intron, a DNA sequence of a gene encoding forphosphinothricin acetyl transferase, and a terminator functional inplants, wherein the two cassettes are transcribed in the same direction,wherein the nucleic acid construct is incorporated into the seed'sgenome on chromosome 8, near position 117, between markers Z1B3 andUMC150a.
 8. A maize plant according to claim 7, wherein the firstexpression cassette comprises SEQ ID Nos. 1-4 in operable sequence.
 9. Amaize plant according to claim 7, wherein the second expression cassettecomprises SEQ ID Nos. 5-8 in operable sequence.
 10. A maize plantaccording to claim 7, wherein the first expression cassette comprisesSEQ ID Nos. 1-4 in operable sequence and the second expression cassettecomprises SEQ ID Nos. 5-8 in operable sequence.
 11. Pollen of the plantof claim
 6. 12. An ovule of the plant of claim
 6. 13. A maize plant, orparts thereof, having all the genotypic and phenotypic characteristicsof a plant according to claim
 6. 14. Hybrid maize seed produced bycrossing a plant according to claim 6 with an inbred maize plant havinga different genotype.
 15. Hybrid maize plant produced by growing hybridmaize seed of claim
 14. 16. A method of producing hybrid maize seedscomprising the following steps: (a) planting seeds of a first inbredmaize line according to claim 1 and seeds of a second inbred line havinga different genotype; (b) cultivating maize plants resulting from saidplanting until time of flowering; (c ) emasculating said flowers ofplants of one of the maize inbred lines; (d) allowing pollination of theother inbred line to occur, and (e) harvesting the hybrid seeds producedthereby.
 17. Hybrids seed produced by the method of claim
 16. 18. Hybridmaize plant produced by growing hybrid maize seed of claim 17.